Identification and characterization of endothelial glycoprotein Ib using viper venom proteins modulating cell adhesion

The expression and function of a glycoprotein Ib (GPIb) complex on human um bilical vein endothelial cells (HUVECs) is still a matter of controversy. W e characterized HUVEC GPIb using viper venom proteins: alboaggregins A and B, echicetin, botrocetin, and echistatin. Echicetin is an antagonist, and a lboaggregins act as agonists of the platelet GPIb complex. Botrocetin is a venom protein that alters von Willebrand factor (vWF) conformation and incr eases its binding affinity for the GPIb complex. Echistatin is a disintegri n that blocks alpha v beta 3. Echistatin, but not echicetin, inhibited the adhesion to vWF of Chinese hamster ovary (CHO) cells transfected with alpha v beta 3. We found the following: (1) Binding of monoclonal antibodies aga inst GPIb alpha to HUVECs was moderately increased after stimulation with c ytokines and phorbol ester. Echicetin demonstrated an inhibitory effect. (2 ) Both echicetin and echistatin, an alpha v beta 3 antagonist, inhibited th e adhesion of HUVECs to immobilized vWF in a dose-dependent manner. The inh ibitory effect was additive when both proteins were used together. (3) Botr ocetin potentiated the adhesion of HUVECs to vWF, and this effect was compl etely abolished by echicetin, but not by echistatin. (4) CHO cells expressi ng GPIb alpha beta/IX adhered to vWF (in the presence of botrocetin) and to alboaggregins; GPIb alpha was required for this reaction. Echicetin, but n ot echistatin, inhibited the adhesion of cells transfected with GPIb alpha beta/IX to immobilized vWF. (5) HUVECs adhered strongly to immobilized vWF and alboaggregins with extensive spreading, which was inhibited by LJ1b1. a monoclonal antibody against GPIb. The purified alpha v beta 3 receptor did not interact with the alboaggregins, thereby excluding the contribution of alpha v beta 3 in inducing HUVEC spreading on alboaggregins. In conclusion , our data confirm the presence of a functional GPIb complex expressed on H UVECs in low density. This complex may mediate HUVEC adhesion and spreading on immobilized vWF and alboaggregins. (C) 1999 by The American Society of Hematology.